CA2541689A1 - Method for the rapid detection of micro-organisms on dna chips - Google Patents

Abstract

The present invention relates to a rapid method of analyzing a population microbial environment, including a microbial population taken from the air. In particular, the method used includes the use of microarrays, with a radioactive detection method or chemiluminescent.

Description

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METHOD FOR RAPID DETECTION OF MICROORGANISMS
ON DNA CHIPS
The present invention relates to a rapid method of analyzing a microbial population in a given environment, including a s microbial population taken from the air. In particular, the method used includes the use of DNA chips, with a method of radioactive or chemiluminescent detection.
The majority of detection methods used today are based on the analysis of DNA or RNA and include the implementation of the 1o PCR amplification technique described in US Patents 4,683,195 and 4,683,202.
PCR makes it possible to amplify target sequences of nucleic acids which are then detected by conventional detection methods.
However, the implementation of an amplification reaction requires 1s about 2 hours.
Other techniques are described in the state of the art, including hybridization methods such as Northern Blot or Southern Blot, using probes, or microplate techniques such as the ELiSA test. These techniques are tricky to implement and 2o relatively expensive, In particular, a large number of microplates and a very large sample volumes would be required for the analysis of several different pathogens in parallel in microplates.
Recently, hybridization techniques have been described on supports solid called chips (US 6,458,584). Such chips are for example As prepared by parallel synthesis of oligonucleotides (Matson et al., Anal Biochem., 1995, 224, 110-116), attached to the surface of a glass support (Ghu et al., 1994, Nucl Acids Res 22, 5456-5465),

2 polypropylene (Matson et al., supra 1995) or gel (Khrapko et al., 1991, J. DNA Sequencing 1: 375-388).
A large number of hybridizations can be performed in parallel using a small amount of sample. However, the hybridization time remains s long enough, usually between 16 and 24 hours.
Despite the existence of many methods available, none allows rapid detection, for example less than 10 hours, even less than 3 hours. However, in the case of Bacillus anthracis infection, the early allocation of an antibiotic is decisive in limiting the 1o mortality. Rapid detection is therefore sought in the case contamination by biological weapons, in order to save the greatest number of lives.
Many species of microorganisms may occur under form of spores, which remain dormant in response to stress 1s environmental. The process during which dormant bacteria transform into vegetative cells is known as sprouting.
Among the bacteria capable of sporulating, mention will for example Bacillus anthracis, Baccillus cereus, Clostridium botulinum abd Clostridiun Perfringens, Vibrio cholera, Vibrio vulnificus, Micrococcus luteus, ao Micrococcus fuberculosis and Legionella pneumophila.
In order to detect bacteria present in the sample in a form sporulated, their cultivation for a period of at least 24 hours is in general recommendation. However, the cultivation for germination of sporulated forms extends the detection time accordingly. In addition, the Culture requires the use of sterile conditions.
To the knowledge of the plaintiff, it has never been described in the state of the technique a rapid method of detecting microorganisms, sufficiently sensitive and specific.

3 There has never been described a rapid method for detecting organisms for the detection of bacterial spores.
Finally, to the knowledge of the plaintiff, no method of detection by chemiluminescence on a sufficiently sensitive DNA chip, and s avoiding too much light scattering on the surface of the chip has summer described.
The invention aims precisely at a method for the rapid detection of micro-organizations of interest in a sample, by molecular hybridization on DNA chips, specific RNAs of these organisms. These RNAs were, in a first step, retro-transcribed with complementary DNA (cDNA), thus ensuring the marking of the sample probes and allowing two possible detection modes on DNA chips, namely a radioactive and chemuuminescent mode. All stages of the method according to the invention is automatable and allows from the collection 1s of the sample to obtain an identification result in 2:30 for the fashion radioactive, and in a little over 3 hours for the chemiluminescent mode.
For the development of a global chain of rapid identification, the invention results in particular from a very significant reduction of the time hybridization. Indeed, it is generally recommended in the literature 2o a hybridization time of at least 16 hours. This time has been reduced to 30 minutes by taking advantage of the kinetics properties of the hybridization reaction.
Indeed, the reaction speed allowing the formation of probe duplexes sample (cDNA) / capture probe (immobilized on the chip) depends Zs mainly the size of these duplexes. So, using probes of small immobilized captures (oligonucleotides from 30 to 90 seas, preferably 70 seas), it was found that the hybridization time could be greatly reduced.

4 Therefore, the present invention provides a method of detection rapid microorganism, comprising:
(a) the collection of micro-organisms, (b) the extraction of the RNA from the microorganisms, s (c) RNA labeling or conversion to complementary DNA
Mark, .
(d) hybridization of labeled complementary DNA or labeled RNA
obtained in step (c) using capture probes placed on a chip and specific for each microorganism, characterized in that the hybridization is carried out in a time of between 15 and 45 minutes, preferably about 30 minutes, (e) detection of possible hybridization, and (f) analysis of the results to determine whether the micro-organisms are present.
The present invention also provides a method for reducing the scattering of light on a chip for mode detection chemiluminescent method, said method comprising (a) the preparation of a chip on the surface of which is deposited a porous membrane impregnated with a chemiluminescent substrate, 2o thus to immobilize the liquid phase of the substrate.
The chip comprising the porous membrane is also part of the invention.
Within the meaning of the invention, the term "environment" includes water, air, plants and soil. Examples of usable environmental samples Zs in the method of the invention are, for example, water, wastewater, soil, mud, waste, sediment, air, etc.
Samples include biological samples taken from the man or the animal. The method is suitable for all types of samples organic. Examples of biological samples include, without being WO 2005/03578

5 PCT / FR2004 / 002558 S
cerebrospinal fluid, lymph, synovial fluid, tears, urine, blood, plasma, serum, skin, tumors, amniotic fluids, tissues, cells and cell cultures.
For the purposes of the invention, the term "target nucleic acid" means an acid s nucleic acid derived from a biological or environmental sample, detected by the capture probe by specific hybridization.
Thus, the target nucleic acid has a sequence that is complementary to the corresponding capture probe and is marked with a marker radioactive or chemiluminescent.
1o The term "probe" refers in the following text to an acid nucleic acid capable of targeting another nucleic acid by pairing complementary bases, including natural bases such as A, G, C, U
or T or modified bases such as ionosine for example. The probe capture is used in the present invention to bind the nucleic acid Is target if such a target is present in the sample.
For the purposes of the invention, the term "micro-organisms" includes all bacteria gram-positive or grain-negative. Bacteria likely to be detected by the process of the invention are preferably pathogenic for humans or animals and / or invasive. The different types of ao bacteria likely to be detected by the methods of the invention, include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus luteus, Micrococcus roseus, Lactococcus lactis, Streptococcus pneumoniae, Streptococcus sludge, Leuconostoc mesenteroides, Oenococcus oeni, Pediococcus damnosus, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhales, Acinetobacter baumannu, Acinetobacter Iwo ~ i, various Citrobacter, Citrobacter freundü, various Citrobacter, Edwardsiella tarda, Enterobacter cloacae, Erwinia chrysantum, Erwinia carotovora, Escherichia coli, Hafnia

6 Alvei, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganu, Pantoea agglomerans, Proteus mirabilis, Proteus vulgaris, Providencia aicalifaciens, Salmonella enterica subsp, enterica, Salmonella enterica, ParatyphiSerratia marcescens Salmonella, Shigella flexneri, Yersinia s enterocolitica; Yersinia pestis, Vibrio parahaemolyticus, Vibrio cholerae, Hydrophilic Aeromonas, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia Mallein, Burkholderia pseudomallei, Ralstonia pickettu, Alcaligenes faecalis, Acinetobacter baumannum, Acinetobacter Iwof ~ i, Stenotrophomonas maltophilia, Campylobacter jejuni, Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, Bacillus anthracis, Bacillus globigü, Listeria monocytogenes, Lactobacillus casei, Clostridium botulinum, Clostridium perfringens, Mycobacterium bovis BCG, Legionella pneumophila, Bordetella pertussis, Francisella tularensis, Brucella melitensis, Brucella 1s abortus, Brucella suis, Brucella canisCoxiella burnetti, Chlamydia pneumonia. This list is of course not limiting.
More specifically, the present invention provides a method of detection rapid microorganisms. Microorganisms are collected by any suitable means known.
ao For air samples, air collection devices or air samplers can be used, including agar media or porous membrane filters or for recovering microorganisms organisms in a defined liquid medium.
If the samples are in a vegetative form, they are subject to a 2s lysis step and an extraction step as described below.
However, if the samples are in spore form, the samples are subject to a revivification stage. This revivification stage ensures early spore germination and eliminates the need for the extreme difficulty of performing efficient lysis of the sporulated forms. The

7 spore form of bacteria is characterized by a reduction in activity physiological cell, internal dehydration and by training Extremely strong external envelopes. This form confers on bacteria a great resistance to the environmental environment s (heat resistance, resistance to certain chemical and physical agents, etc.).
The transition from the sporulated form to the vegetative form (phenomenon of germination) is triggered by the reinstatement of growth, hence this stage of revivification.
Germination of the spores can be achieved by any known method of the state of the art, for example, by heating the suspension of organisms at 65 ° C for 20 minutes or adjusting the gradient.
osmotic microorganisms by sequential diffusion. (series extractions), progressive diffusion (sequential dilution) and diffusion continuous (dialysis). The method disclosed in US Patent 6,589,771 can 1s also be used. The use of buffers with osmolarity less than that of cells, such as PBS buffer (Dubecco's phosphate Buffered Saline), pH 7.3, 0.9% NaCl is another useful method.
The addition of L-alanine in the culture medium having a pH between 5.5 and 7.0 at a temperature between 4 ° and 55 ° C may also be a, o used for germination of spores, as well as the medium known as the name Brain Heart Infusion Broth, which is a medium especially preferred for germination.
The revivification step includes centrifugation of the liquid sample for 1 to 3 minutes, preferably 2 minutes at 10,000 xg, the 2s resuspension of the pellet in the medium HIB and incubation with stirring for a period of about 30 to 45, preferably 40 minutes to a temperature between 4 ° C and 55 ° C preferably 37 °
vs.
After this stage of germination of the spores, the microorganisms are subjected to lysis and the extraction step, for the extraction of RNA.

8 The objective of this step is to lyse all the bacteria contained in the concentrated sample, after collection, and to extract all of them RNA in as short a time as possible.
In this respect, the protocol requires that microorganisms be lysed by chemically, mechanically or thermally. For a chemical lysis, everything appropriate lysis medium such as zymolysis, cellulose, ~ -glucururonidase, lysostaphin, lysozyme and others can be used in the method of the invention.
Mechanical means for lysis of microorganisms include Homogenization, stirring in the presence of glass beads, sonication, the French press, etc. Thermal lysis consists in heating the samples at a temperature of 80 to 90 ° C. It is better to use a combination of chemical lysis using lysozyme and lysostaphin followed sonication at this stage of this method.
After lysis of the cells, the RNA is extracted from the cells. Any method known from the state of the art can be used for the extraction of RNA
cells, and in particular the numerous RNA extraction kits commercially available. For example, we will use the RNAeasy kit of QIAGEN ~, following the manufacturer's instructions, or any other kit ao extraction whose implementation is simple and fast.
The RNA obtained can be labeled radioactively or with the aid of a marker chemiluminescent. Alternatively, a DNA can be synthesized from of an RNA using reverse transcription methods, said DNA
being labeled during the reverse transcription step by incorporation of 2s modified nucleotide.
This step of synthesis of the probes corresponds to the reverse transcription (retrotranscription) RNAs extracted into labeled complementary DNAs.
Two different types of markers can be used as an alternative

9 in the process of the invention. Target nucleic acids can be radiolabelled or labeled with a marker allowing the revelation in chemiluminescent mode.
Any radioactive marker may be used for this purpose, including 32P, s5s, 1251 ' s P33, Hs, 1311, etc. It is preferable to use 35S.
The principle of chemiluminescent revelation is based on the use enzymes that specifically degrade chemiluminescent substrates.
This enzymatic degradation causes the emission of photons. In this case, the sample probes are marked either directly with the enzyme, are indirectly by a specific molecular complex (biotin / streptavidin / biotin-enzyme complex). These two types of marking of sample probes are shown schematically in Figure 1.
Any enzyme known for its use in chemiluminescence can 1s be used to label the target nucleic acid of this invention.
As examples, alkaline phosphatase, ~ -galactosidase, (i-gluconidase, ~ i-glucosidase, peroxidase, luciferase, etc.
examples, chemiluminescence complexes include biotin, avidin and streptavidin or antibody-antibiotic complexes ao or anti-digoxigenin coupled to the enzyme, filuorescein-antibody antifluorescéine.
In addition to the marked sample, witnesses will be conventionally used also marked radioactively or with the help of a marker chemiluminescent. The control RNAs are characterized by the total absence 2s of hybridization on the capture probes. The cookies used are by RNA encoding tyrosine hydroxylase (TN) or neo RNA encoding resistance gene to neomycin.

The advantages of reading in chemiluminescence include to improve the sensitivity possibility of amplifying .the signal) and perform a quantitative measurement. In addition, the equipment necessary for signal measurement is simpler than for fluorescence measurements s (no excitatory light source) and manipulations are facilitated compared to the use of radioactive products.
The major problem of chemiluminescent revelation lies in the fact that the chemiluminescent substrates are in liquid solution. However, the diffusion photons in the liquid, as well as the movements of the liquid on the flat surface of the chip, cause a significant decrease in the spatial reading resolution. This phenomenon of light scattering makes the measurement of the size of the spot on the. chips to DNA.
The invention consisted in defining a device making it possible to immobilize the Is a liquid phase (chemiluminescent substrate) in contact with the surface of the glass slide, to limit the diffusion of light.
This device consists of a porous membrane impregnated with the substrate.
This membrane is deposited on the surface of the chip, thus creating a three-dimensional network immobilizing the liquid phase. This system allows 2o significantly reduce the photon scattering phenomena, this making compatible the reading with the diameter of the spots.
Figure 2 shows the two flea photographs below, which compare the results of the measurements obtained in the glass slide configurations impregnated membrane and glass slide 2s only.
Before preparing labeled samples of microorganisms, one prepares the chips containing immobilized capture probes. The Capture probes can be DNA or RNA molecules.

The capture probes are preferably chosen firstly from specific regions of the 16S rRNAs of each agent, on the other hand in specific regions of different messenger RNAs. Capture probes corresponding to the 16S rRNAs were chosen in the most s variables of these RNAs, in order to ensure the identification of the strains used.
For capture probes corresponding to mRNAs, one will choose MRNA whose level of expression is well known, in order to represent better the different physiological states possible for agents collected. Have been selected, mRNAs encoding proteins 1o involved: in the germination of spores; in the cell cycle; in great metabolic functions; in the expression of virulence. With this type of wide choice, it is reasonable to be able to get signals regardless of the physiological state of the agent collected, knowing that a revivification step is carried out before the preparation of the probe Is sample.
The size of the capture probes can vary from 30 to 90 nucleotides, from preferably about 70 nucleotides. Usable probes can carry a amino group at their 5 'end.
For the manufacture of chips, a thin layer of glass is used, 2o covered with polylysine, silane or epoxy. In a mode of preferred embodiment, the epoxy used is 3-glycidoxylpropyl trimethoxy silane. This type of epoxy reacts spontaneously in the presence of water with the 5 'amine end of the capture probe and allows a binding covalent at the 5 'end of the probe. This specific link leaves the as remaining part of the free probe for hybridization.
The different nucleotides which are aminated at the 5 'end are deposited on the glass plate using a "spotter". For example, the diameter average spots can be between 10 and 150 pm The distance between the two spots, from one center to another can be between 300 and 500 p, m.
The sample probes (DNA or RNA) previously labeled are deposited on the surface of the chip and will hybridize by homology of s sequence at the corresponding spots.
The hybridization is carried out in about 30 minutes. After this phase of hybridization, the chip is washed and the reading reveals the effective hybridizations.
The protocol for the hybridization step is identical depending on the type of 1o labeling done, radioactive or chemiluminescent. ' Primarily, nucleic acid probes are first denatured and a hybridization buffer is added to the denatured nucleic acid. The mixture is then placed on the chip containing the capture probes.
The chip is then placed in a thermostatically controlled chamber at 42 ° C.
and is allowed to incubate for about 30 minutes. The chip is then removed from the enclosure and washed.
Depending on the type of marking, radioactive or chemiluminescent, the method of revelation is different. If radioactivity is used, the chip is covered solid scintillant and the analysis is carried out by means of a device ao adapted (phosphorimager, car radio-graphic ...). If a marker chemiluminescent is used, a thin nylon membrane is impregnated a chemiluminescent substrate, which is diluted in a suitable substrate and placed on the chip. The revelation is then done using the micro-imager.

EXAMPLES
4 model strains representing grain - and grain + bacteria under vegetative or sporulated forms were used. These model strains correspond to Escherichia coli (grain -), to Pantoa agglomerans (grain -, s CIP 82 100), with Bacillus thuringiensis, subspecies kurstaki, in the form vegetative (Grain +, CIP 105 674) and Bacillus globigü in the form sporulated (CIP 77 18). To identify these four strains, we pulled part of the molecular data available in the banks of data, by choosing capture probes immobilized on the chip corresponding to both variable regions of 16S ribosomal RNAs (16S rRNA) and specific messenger RNAs (mRNAs). In a context broader, the joint detection of these two classes of RNA
identification, thus giving access to the genus and the species.
Example 1: Preparation of the sample 1s A. Revivification protocol This step consists in incubating the biological agents recovered in the collection medium in 200 μl of revivification medium.
The collection medium containing the strains is centrifuged for 2 minutes to 10,000 x g.
zo The bacterial pellet is taken up in 200 NI of HIB revivification medium (Brain Heart Broth Infusion, ICN, Ref: 1002417), and incubated under stirring for 40 min at 37 ° C.
B. Lyse and Exfraction The cultures are centrifuged in the revivification medium for 2 2 minutes to 10,000 x g.

The bacterial pellet is then taken up in 50 NI of lysozyme at 12 mg / ml (solution diluted in TE) (Sigma), 50 NI lysostaphin 100 μg / ml (diluted solution in TE) (Sigma), and finally carried out sonication of the samples for 30 seconds at 45 Hz.
The incubation is then carried out for 10 minutes at 37.degree.
there adds 350 μl of RLT-2ME buffer (RNAeasy kit from Qiagen), as well as 250 μl of absolute ethanol, which is mixed by suction / discharge and that is deposited on the RNAeasy column (absorption column). The whole thing is then centrifuged at 8000 xg for 15 sec.
The column is then washed with 700 μl of buffer RW.1 (centrifugation at 8000 xg for 15 sec) and then with 500 μl of RPE buffer (twice) (Centrifugation at 8000 xg for 15 sec).
The column is then centrifuged for 2 minutes after the last wash to remove excess wash buffer.
1 μl of sterile water is then added to the center of the column and the RNA
are eluted by centrifugation at 8000 xg for 1 min.
If necessary, the RNAs are stored at -80 ° C.
C. Synthesis of sample probes Two different protocols are possible.
zo Protocol for detection in radioactive mode pL of pdNs (50 ng / μl) are added to 7 μl of the RNA mixture containing the total RNA extracted previously, and 5 ng tyrosine control RNAs hydroxylase (TH) and Neo.
The mixture is then incubated for 5 min at 70 ° C. and then cooled.
quickly 2s at 4 ° C.

The following reagents are then added at 4 ° C: 5 ~, I buffer RT 10 X,

10 p, 1 MgCl 2 (25 mM), 5 μl DTT (100 mM), 2.5 μl RNasin (40 U / p, L), 1 ~, I mixture dCGT-TP (25 mM each), 5 ~ I dATP 35S (1000Ci / mmol; 10 ~, Cil ~ L), 2.5 p, 1 ddTTP (1 mM), 2 p1 Super Script II (200 U / N!).
The mixture should then incubate for 2 min at 25 ° C and then 30 min at 42 ° C.
Stopping the reaction and hydrolysis of the RNAs is ensured by the addition of 7.5 p1 of 2M NaOH (alkaline hydrolysis), incubation for 5 min at 50 °, the addition of 7.5 μl of 2.2 M acetic acid (neutralization) and finally 1o purification of the P10 exclusion column probes (Biô-Rad). Volume , final elution is 40 p1.
Protocol for chemiluminescent detection 10 μl of pdN6 (50 ng / μl) are added to 7 μl of the RNA mixture containing previously extracted total RNAs and 5 ng TH control RNAs and is neo. The mixture is then incubated for 5 min at 70 ° C. and then cooled.
quickly at 4 ° C.
Always at 4 °, the following reagents are added: 5 μl of buffer RT 10 X, 10 ~, I of MgCl2 (25 mM), 5 ~ I of DTT (100mM), 2.5 p, 1 of RNasin (40 U / ~ I), 1 ~, I of dCGA-TP mixture (25 mM each), 5 μl of dUTP-biotin (1 mM), ao 1 p1 of dTTP (10 mM), 1 ~ I of 35S dATP (1000Ci / mmol; 10p, Ci / p, 1), 2.5 ~, I
ddTTP (1 mM) and 2 ~ .I Super Script II (200 U / pl).
The mixture is then incubated for 2 minutes at 25.degree.
min to 42 ° C.
Stopping the reaction and hydrolysis of the RNAs is ensured by the addition of 7.5 2M NaOH (alkaline hydrolysis), incubation for 5 min.
50 ° C
and the addition of 7.5 μl of 2.2 M acetic acid (neutralization).

The probes are then purified on an exclusion column P10 (Bio-Rad) the final elution volume is 40 p1.
Example 2: Capture probes A. Presentation of witnesses s As a control on the chip, we use capture probes corresponding to TH and neo RNAs. These RNAs are directly added to RNA extracts just before the synthesis of the probes samples. 11s thus allow, on the one hand, to control the efficiency of the synthesis of Labeled cDNA, on the other hand to ensure the column rows markings on the io chip.
TN:
GGGACATGTACCCATGTTGGCTGACCGCACATTTGCCCAGTTCTCC
CAGGACATTGGACTTGCATCTCTG (SEQ ID No. 1) Neo:
is AGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCAT
CATGGCTGATGCAATGCGGCGGCT (SEQ ID No. 2) B. Choice of capture probes corresponding to 16S rRNA
The sequences of these capture probes are as follows 20 16Scoli GTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCA
TTGACGTTACCCGCAGAAGAAGCACC (SEQ ID No. 3) 16Spa AAAGTACTTTCAGCGGGAGGAGGCGATGGGTTAATAACCTGTCGAT
~ s TGACGTTACCGCAGAAGAAGCACC (SEQ ID No. 4) 16SBt1 CATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTC
ACTTATGGATGGACCCGCGTCGCA (SEQ ID No. 5) l6SBt2 s GATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACT
GCATGGTTCGAAATTGAAAGGCGG (SEQ ID NO: 6) 16SBt3 CGG GAAACCG GGG CTAATACCG GATAACATTTTGAACTG CATG GTT
CGAAATTGAAAGGCGGCTTCGGCT (SEQ ID NO. 7) io 16SBS1:
TAATAC CG GATG GTTGTTTGAAC CG CATG GTTCAAACATAAAAG G TG
GCTTCGGCTACCACTTCACGATG (SEQ ID No. 8) TGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGT
! s GCCGTTCGAATAGGGCGGTACCTT (SEQ ID No. 9) For the ARN16S capture probes, all cross hybridizations possible from one agent to another is well known and can be summarized in the mismatch table below.
E.coli Bth Pagg B. Globig 16Scoli 0/70 31/70 13/70 38/70 16SBth1 40/70 0/70 35/70 16/70 16SBth2 32/70 0/70 28/70 11/70 16Bth3 33/70 0/70 29/70 11/70 ~

16SPagg 11/70 33/70 0/70 39/70 C. Choice of capture probes corresponding to ARNm-Capture probes corresponding to specific mRNAs of B. thuringiensis:
s 1. Specific cryogenic gene of B. thuringiensis:
cry (accession number: AF278797) is a plasmid gene expressed only during the stationary phase of the cell growth cycle.
This gene produces a very effective endotoxin against a large number of insects.
1o Oligonucleotides (70 seas) BTCY1 CGCTTGAAAGTTGGGTTGGAAATCGTAAGAACACAAGGGCTAGGAGT
GTTGTCAAGAGCCAATATATCGC (SEQ ID NO.10) 2. Specific inhA2 gene of B. thuringiensis:
The inhA2 gene (accession number: AF421888) is described in the article is next: Fedhila, S., Nel, P, and Lereclus, D. 2002. The InhA2 metalloprotease of Bacillus thuringiensis strain 407 is required for Pathogenicity in infected insects via the oral route. J. Bacteriol. 184 (12), 3296-3304.
The inhA gene produces a zinc-dependent metalloprotease. This protein ao hydrolyzes the cecropins and the attachments involved in the system of humoral defense of insects. InhA expression starts at the beginning sporulation and is indirectly activated by SpoA protein. The gene inhA2 produces a protein that has 66.2% identities with the protein InhA and which contains a zinc binding site. InhA2 protein plays a role 2s important in virulence. The inhA2 gene is expressed when the bacteria are growing or sporulating in a rich medium (such as the revivification medium), whereas the inhA gene would preferentially expressed when the cells are in a poor environment.
Oligonucleotides (70 seas) BTinh1 GCAAGTTCCAACTTCAAAAGCAAAGCAAGCTCCATATAAAGGATCTGT
s TCGATCGGATAAAGTATTAGTG (SEQ 1D NO.11) 3. Specific hblA gene of B. thurinqlensis, B. cereus and Edwarsiella tarda:
The hblA genes (accession number: AJ243149, AJ243147, AJ007794, AJ237785, AJ243163, L43071) are described in the following:
Pruss, BM, Dietrich, R., Nibier, B., Martlbauer, E. and Scherer, S. 1999. The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group. Appl. About. Microbiol. 65 (12), 5436-5442 (1999) Okstad, OA, Gominet, M., PurneIle, B., Rose, M., LerecIus, D. and Kolstø, AB1999. Sequence analysis of Bacillus cereus loci carrying is PIcR-regulated genes encoding degradative enzymes and enterotoxin.
Microbiology 145 (Pt 11), 3129-3138.
Chen, JD, Lai, SY and Huang, SL 1996. Molecular cloning, characterization, and sequencing of the hemolysin gene from Edwardsiella soon. Arch. Microbiol. 165 (1), 9-17 ao The hblA gene produces an enterotoxin (hemolysin BL) that binds to HBL complex.
Oligonucleotides (70 seas) HblA1 GGATACGAAAGTAAAAAAACAGCTTTTAGATACATTGAATGGTATTGTT
2s GAATACGATACAACATTTGAC (SEQ ID No. 12) 4. Specified gyrB gene of group B cereus ~
The gyrB gene (accession number: AF090331, AF136390, AF136387, AF090332, AF136388) is described in the following article Yamada, S., Ohashi, E., Agata, N. and Venkateswaran, K. 1999. Cloning and s nucleotide sequence analysis of gyrB of Bacillus cereus, B. thuringiensis, B.
mycoides, and B, anthracis and their application to the detection of B.
cereus in rice. Appl. About. Microbiol. 65 (4), 1483-1490.
GyrB is a subunit protein of DNA gyrase (topoisomerase) type II). It allows the reduction of the mechanical stresses affecting 1o the double-stranded DNA during the replication and transcription steps.
Oligonucleotide (70 seas) GyrB 1 CGTAATATTAAATTAACAATTGAAGATAAACGTGAACATAAGCAAAAGA
AAGAGTTCCACTATGAAGGTGG (SEQ ID NO: 13) 1s D. Capture probes corresponding to mRNAs specific to B. globigü:
1. Specified recA gene of B cereus, B anthracis and B_ subtilis:
The recA gene (accession number: AF229167, AY147925) is described in ao following articles: Ko, M., Choi, H. and Park, C. 2002. Group I self splicing intron in the recA gene of Bacillus anthracis J. Bacteriol. 184 (14), 3917-3922; Maughan, H., Birky, CW Jr., Nicholson, WL, Rosenzweig, WD and Vreeland, RH The paradox of the ancient bacterium which contains 'modern' protein-coding genes Unpublished.

RecA is a recombinase of type A, it repairs by recombination the DNA replication errors.
Oligonucleotides RecA1: specific for the group B. cereus and B. anthracis (groups close to 8. thuringiensis ~ and RecA2: specific to B. subtilis s of which 8. Globigü is a subspecies.
oligonucleotides RecAl: specific for B. cereus and B. anthracis GGTGGTCGTGCGTTGAAATTCTATTCAACTGTTCGTCTTGAAGTGCGT
CGTGCGGAGCAATTAAAACAAG (SEQ ID No. 14) 1o RecA2: specific of 8. subtilis CGACGTGAAGCAGAATGTGCTTGGTGTCAAGAAGGAAACATTAGATCG
TTTTGGGGCGGTCAGCAAGGAG (SEQ ID No. 15) 1. B.subtilis specific n17D gene:
The n97D gene (accession number: D31856) is described in the following article:
is Oda, M, Sugishita, A. and Furukawa, K. 1988. Cloning and nucleotide sequences of histidase and regulatory genes in Bacillus subtilis hut operon and positive regulation of the operon. J. Bacteriol. 170 (7), 3199-3205.
The n97D gene produces an enzyme, 6-phospho-1-glucosidase involved 2o in glucose metabolism.
Oligonucleotide (70 Seas) N17D
CGCAAAACACTTTTCTTCTCAGATGTTCAGGCAAGAGGCGCATACCCG
GGATATATGAAACGCTATCTGG (SEQ ID No. 16) 2. Specified gpr gene of B. subtilis;
The gpr gene (accession number: 299117) is described in the following article:
Nessi, C., Jedrzejas, MJ, and Setlow, P.1998. Structure and mechanism of the protease that degrades small, acid-soluble spore proteins during germination of spores of Bacillus species. J. Bacteriol. 180 (19):
5077-5084.
The gpr gene is specific for 8. subtilis. This gene produces a protease active during germination of spores. It degrades the DNA associated with small soluble proteins (SASPs).
o Oligonucleotide (70 seas) gpr1 GGCACTTGTTTAAGCTTCAGCCGGAAAACGTACAGGAGGGTTACAGG
CCTGTCAGTGCTTTTGCACCGGG (SEQ ID NO.17) 3. B.subtilis specific sleB gene:
The sleB (accession number: 299117) gene is described in the following article Moir, A., Corfe, BM, and Behravan, J.2002. Spore germination. Cell. Mol.
Life Sci.59: 403-409.
The sle8 gene is specific for B. subtilis. This gene is involved in the early stage of spore germination. It is a lytic enzyme that degrades the spore cortex.
ao Oligonucleotide (70 seas) sIeB1 GGGAATACATTCACGCATTACGGAAAAATTCCGCTAAAGTATCAGACG
AAACCATCAAAAGCAGCAACAC (SEQ ID NO.18) 4. CvirlD gene of B. subtilis:
The cvvlD gene (accession number: 299117) is described in the following article Moir, A., Corfe, BM, and Behravan, J. 2002. Spore germination. Cell. Mol.
Life Sci.59: 403-409. The cwlD gene is specific for 8. subtilis. This gene It is involved in the early stage of spore germination. It's a hydrolase which degrades the wall of spores. His product is N-acetylmuramoyl-L-alanineamidase.
Oligonucleotide (70 seas) cwlD1 GGCTATAGCCGACGAAAAGCTGAGGATCTAAGACAACGAGTCAAATTA
ATAAACCATTCAGAGGCGGAGC (SEQ ID NO.19) 5. Specific gene of B. subtilis:
The cwlJ gene (accession number: 299117) is described in the following article Moir, A., Corfe, BM, and Behravan, J. 2002. Spore germination. Cell. Mol.
Life Sci. 59: 403-409.
1s The cwlJ gene is specific for 8 subtilis. This gene is involved in the early stage of spore germination. It is a hydrolase that degrades the wall of spores.
Oligonucleotide (70 seas) cwIJ1 CGCGTTTGAGGCTGTGACTCATGGATATTTTTATCAAAGGGCGCGAGA
ao TAGCGAGCGTGCCCTTGCACGC (SEQ ID NO.20) 6. Specific dnaG gene of B, subtilis:
The dnaG gene (accession number: Z99117) is described in the following article Kunst, F., et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis Nature 390 (6657), 249-256.
as The dnaG gene encodes a primase intervening at the beginning of the replication of DNA.

Oligonucleotide (70 seas) DnaGBS
GGCCCGCCTTCATATCAGAAAGCAGGAAAGAGCAGTCTTATTTGAAGG
GTTTGCTGATGTCTATACGGCC (SEQ ID NO: 21) E. Capture probes corresponding to mRNAs s specifics of P. agglomerans:
1. Specific bglA gene of P, agglomerans:
The bglA gene (accession number: X79911) is described in the following article:
Marri, L., Valentini, S. and Venditti, D. 1995. Cloning and nucleotide sequence of the bglA gene from Ervvinia herbicola and expression of beta-Glucosidase activity in Escherichia coli. FEMS Microbiol. Lett. 128 (2), 138.
BgIA is a phospho- ~ 3-glucosidase A, it hydrolyzes cellulose.
Oligonucleotide (70 seas) BgIA1 GGTTGACCAGTTTGTACTGATTAATGAACCAACCGTTGAAGTTGCAAC
is AAAAATCATGGCCGAGAAACGA (SEQ ID NO 22) 2. Specific tufB gene of P. agglomerans:
The tutB gene (accession number: AF418598, U25347) is described in following articles: Katayama, T., Suzuki, H., Koyanagi, T. and Kumagai, H.
2002. Functional analysis of the Enwinia herbicola tutB gene and its ao product. J. Bacteriol. 184 (11), 3135-3141. Foor, M. 1995. Nucleotide and genes of Ervvinia herbicola.
Unpublished tutB is a tyrosine permease (tyrosine transporter) and participates in metabolism of tyrosine.

Oligonucleotide (70 seas) tut1 GCTACCTTCTCTGGCCTGATATGGCATGTCGAAGGCGCTAAACTTATT
GACAGTGCCGCCTGGGCGCTGC (SEQ ID NO.23) 3. specific ipdC gene of P aaalomerans ~
s The ipdC (accession number: L80006) gene is described in the following article Brandi, MT, Quinones, B., and Lindow, SE 2001. Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces. PNAS 98 (6): 3454-3459.
ipdC produces an enzyme indol-pyruvate decarboxylase involved in the lo metabolism of a hormone of plant origin. Expression of the ipdC gene is induced by plants but also by other conditions as drought conditions.
Oligonucleotide (70 seas) ipdc1 CGATCTGCAGCCCTTCAGTGCCAGCGTGGGTAATGAACGTTTTGCGC
CGTTGTCGATGGCGGATGCGCTC (SEQ ID NO: 24) 4. Specific crtH gene of P agalomerans ~
The gene crtH (accession number: L17139, M87280) is described in the article next HundIe, B., Alberti, M., Nievelstein, V., Beyer, P., Kleinig, H., Armstrong, GA, ~ o Burke, DH and Hearst, JE 1994. Functional .assignment of Enivinia herbicola Eho10 carotenoid genes expressed in Escherichia coli. Mol. Gen.
Broom. 245 (4), 406-416.
This gene allows the synthesis of β-carotene.
Oligonucleotide crtH1 CCCGCGCAAGGGCGTATTTGAGCTAAACGATCTCTTTGCGGTGGTGTT
TGCCGGGGTGGCTATCGCGCTG (SEQ ID NO.25) F. Capture probes corresponding to mRNAs specific to E. Coli 1. Specific pgm gene of E. colin The pgm gene is described in the following article WeIch, RA, et al. 2002. Extensive Mosiac Structure Revealed by the Complete Genome Sequence of Uropathogenic Escherichia coli. PNAS 99 (26), 17020-17024.
This gene produces an enzyme that participates in both degradation and glucose synthesis. It belongs to the family of phosphohexose Mutases.
Oligonucleotide (70 seas) pgml GGCGGTTCCGGTATCGAATACTGGAAGGGTATTGGCGAGTATTACAAC
CTCAACCTGACTATCGTTAACG (SEQ ID NO: 26) 2. IipB specific gene of E. coli ~
1s The IipB gene (accession number: AE016757.1 ~) is described in the article next: Sean W. Jordan and John E. Cronan, Jr. 2003. The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl) -Acyl Carrier Protein: Protein Transferase. J. Bacteriol. 185 (5): 1582-1589.
LipB is involved in the attachment of the lipoyl group to proteins in 2o creating an amide bridge. This protein joins the carboxyl group of the acid lipoic groups ~ -amines lysine residues.
Oligonucleotide (70 seas) LipB1 GGGGCAACAGGTGATGTATGTGTTGCTTAACCTGAAACGCCGTAAACT
CGGTGTGCGTGAACTGGTGACC (SEQ ID NO 27) 3. Specific dnaG gene of E. coli:
The dnaG (accession number: V00274) gene is described in the following article Smiley, BL, and a1.1982. Sequences of the Escherichia coli dnaG primase gene and regulation of its expression. PNAS. 79 (15), 4550-4554.
s The dnaG gene encodes a primase intervening at the beginning of the replication of DNA.
Oligonucleotide (70 seas) DnaG1 CGGGTGATTGGTTTTGGCGGGCGCGTGCTGGGCAACGATACCCCCAA
ATACCTGAACTCGCCGGAAACAG (SEQ ID NO: 28) 1o Example 3- Manufacture of chips For the manufacture of chips, we use epoxy glass blades, QMT Epoxy slides (Quantifoil # S100005). The epoxy blades used are coated with 3-glycidoxypropyl trimethoxy silane.
The various 5 'amino oligonucleotides (Eurogentec) are diluted to 10 1s or 15 μM in QMT solution spotting Solution I recommended by Quantifoil (QMT Epoxy Kit # S106010, Quantifoil). Capture probes are deposited on the glass slides using the "spotteur ~> QARRAY
(Genetix) with full needles (Proteigene). The average diameter of spot is 200 pm and the inter-spot distance center to center is Zo 500 pm. Once the deposit is made, the covalent bond between the function amine capture probes and the epoxy support is performed by placing the blades in an enclosure with a saturated humidity atmosphere for 30 min at 37 ° C. The ready-made chips are air-dried and kept at room temperature away from moisture until use.
2s Just before proceeding to the hybridization step, the fleas are treated following the protocol recommended by Quantifoil: They must incubate 5 minutes in the washing solution I at room temperature, incubate twice 2 minutes in the washing solution It at room temperature, incubate 10 minutes in washing solution III at room temperature, incubate 1 minutes in dH20 at room temperature, incubate 15 minutes in 1X
QMT Blocking solution at 50 ° C and finally incubate 1 minute in H20 at s room temperature.
Presentation of a chip plan The position of the deposits of the capture probes on the DNA chip is shown in the diagram below in Table 1 . ~~ ,. . _. , i. ~ '-......: t, l ~ ll 'I, I, 1,1' ~~~ e ...
'v.v'W:' ~ '';'~' ° ~ 1 1 1 1 1 ~ 1 1 1: ~~: ~: ~ ':.
. ;. ~ A .; ;.; :. .Lli ..: ... 1, 1 I 1,, 1 1 ~ II 1 1 ~:
. ... f ~ xs.: ...: 1 I 1 ~, 1, 1 I 1 I, 1, ~ ... ~ ~ "" ~~
1, 1 1 1 1 1, I 11 the 1; ~ ...
.,; ~. ... the 1 II 1 1 I ((~~, 1 I 1; "c ;; ..: ..; I y, I 1 .? ... 1,111,, I, ~ 1I11: ....._'...
.: ...: 1 I 1 1 1 1:.
~. ~ ..: 1 II, II, II Ilffl I, 1 .. ~ v ~~ ° ~.
~: 'a3:': ~: '~~, ~ I 1' the 'l' the 'l'.
~ .. ~ .... ~. ..; ...;. . . , .....
.. , Ie!' I 1; ~ ...
: ~: i ~ ':': ~: ~: ~: ~:, T,: ~:.: ° ..: ..., 1, ~ t 11, ...; .., ... '"
,. ~. ~ ~; a:. . . ~ .. ~ ..., I, ~ 1, 1,: ...; ._ ::: ':
': ~: ~ 3: ~:: ~': ~:: ~. . ~: '' . ,:.: .. r .... 1 1 1 ~,?; ' ~: ~: ~! rt: ': ~:' .. Itl II, ~ ...
::, ~, ~ 1, ~, :: .......
, III. ~ '~. : ~ ::: ..
': ~: 7s: ~:: ~:: ~~:: ~:' ~.:.:, ~ .._ .., 1 I,:.
~. ~ 'shot:' ~. : '-.y' ~. ~ ~~: ~ 'L ~~;; ..., 1 I 1 1 I; ~ .... ~: .....
1 ::
.., 1, ~, II: ~ ..
~ '~: ~; '~' ~ '~' ~. ... .. .. .. ...::. , ~ ' ~: the t ~ l '! ~ 1 ~ 1 II .. ~. ' . 1 'the I
:. d,. ~. ~ r, 1 p W tt '' ~~ '' ~, ~ ... ~ ... iy ~ °. ~ ..... 1 ~ I ~ I ~ 1 N
: ~: Ui:: ~: I 1y ~ 1 IIII ~ 0v1 I. ...: C: ...: .. IIII p ~ :: '!'. ':: ~:, I ~ I ~ I ~ t 1 ~ 1 ~~ 1 ~ ..: .. 1 ~ 1 ~ 1 ~ 1 ..., t 1 tt 1 I ~:: t 1 1 1 ; I 1 II 1 II ~~ I. ...; :. 1 1 ~ 1 I 'I'l' 111 111 III ~. I. ~~~. 1 IIIII
ril I .....; ~ ... ~. II ~ III 1 : ~: 3 ~: ~ :; ~, I 1 1 1 f <~, p 1. ... IQX ... i. I 1 '! -I 1 1 1 I 1 I 1 I ~ '~' 1 I 1 1 1 1 . ~ 1 1 I 1! I ~ I. ...: ~ eT ...: .. 1 1 IIIL
t 1 1 (, ~ I 1 "1 1 I 1 1 I
. ~ ':' 1 1 1 1 1 1 t. :. II 1 I 1 I
1 1 1 1 II 1, ~~. . . ~ I 1 I 1 1 1 1 .:. . ~. II 1,11 111,111,1:.: ..., 1111 ; ...; : ~: i ~ t:: I, II 1 I, 1 I ~ J.,, 1: 'i ~ ~, ...: ...;
: ~: I f1 1 III t I ...? .. ~ ...:.
1 II 1 II ..
. ~; , 1; ~:
:, ~ II 'I' I '1 II ~ 1 ~, ~~.
(B '...' ~.: ~ 'Y';~;';. 1 ~ the ~ T' ~ 1 ~ II 1 ~ II 1, ...
1 ~~ I 1 ~ I ~~ 'I ~,:. ~ ..? C ~ .... t0 ... ~ wi ....; ~:: ~ 1 I 1 .I 1 II 1 I, ~ I 1 I:. '?' ° ~ .. ".. ~
: ~: L: ~:::. 1, 1 sJ, I, II 1 1 II 1 ~~:
: ~.: .., i1 ~ 11; I ~ ::
I ',' 1 '::. :: ~ :: ~' ~
PS::: ~~ .ï, ~.: ...,, 1 1:.;.
. .fin. . ..., ~ re .... 1 I,, '1: ~ ..ò .F .- ..: ...:.
: ':, 1 "' ~ ':: 1 I ~ .. ....
1, I tf ~ l 1 II ~ '' : ': r:' :: ~ ~. .... I ~ III, I:. . .i .. ::: ~ ':
': .t ?:.'. '"... ~., ... 1 1 II:. ::
.:; .. ~ y.: ...: I 1 II 1,; ....: ~~ T ~. ~. ~.
': 1 :.'. ':. ...: .., ~.: ...: IIIIII ....
~ The L::. ... 1 ~~ 1 1 1 ~ ° III: ~.
. ~. . . ,: .....-:
", ~ ::.: '' p0 ;. ...: fit ..; ...
..:.
.... d. ï ... ~
:: cr ..: ...
.:. ~;
:: J. ::.
: '. ~ cr::': :::
...
~~
:. ... '0: ...: .. W
. . vy. . : ~ d:
...
':':
~. . ...: ':' G0. '.'. . '. ~~. ':, ~ i ...' ~~ "'? ....?.
.. ::
~, ~:
:. :. ' . ~ .: ': .c.' ::.: ...: ... ~ a ~.; ...:.
'.'. '0 ..'. '.''.'. Pp '::

Example 4 - Hybridization step of the chip This step corresponds to the specific fixation of the sample probes marked on the complementary capture probes (on the chip). She is therefore simply to deposit on the chip the sample probes.
s Depending on the chosen detection mode (chemiluminescence or radioactivity), two different protocols are possible.
A. Protocol for detection in radioactive mode The 40 μl of labeled sample probe are evaporated under vacuum to obtain a final volume of 3.4 p1. The probe is then denatured for 2 10 min at 95 ° C., then added to the ice: 7 μl of 20 × SSC filtered extemporaneously, 20 μl of 100% formamide, 8 μl of 25% dextran sulfate, 0.6 μl of 20% filtered SDS, 1 μl of yeast tRNA at 3 mg / ml.
The entire volume of this solution is then deposited on the region of the chip containing the spots and a piece of parafilm placed on the field of is chip to avoid evaporation of probes during hybridization.
The blade is placed in the hybridization chamber under conditions guaranteeing the absence of evaporation of the sample probe.
The chamber is then placed in a thermostat-controlled enclosure 42 ° C
for 30 minutes. The blade is then removed from the chamber and washed at 42 ° C.
2o according to the following protocol: 2 min. in solution I: SSC 2X / SDS 0,1 %
2 min. in solution II: 1 x SSC and 2 min. in solution III: SSC
0.2X.
The slide is finally dried by centrifugation for 5 minutes. at 980 rpm, at room temperature.

B. Protocol for chemiluminescent detection This protocol is identical to the one presented above. After drying the slide by centrifugation for 5 min at 980 rpm at room temperature, the following steps are added s Blades are exposed to a 0.120 joules UV source to achieve covalent bonds between the hybridized sample probes on the capture probes.
They are then rinsed rapidly in 1 × PBS buffer, then saturated with 400p1 of ABC complex (Vector: Alkaline phosphates 1o standard ABC kit; ref. AK-5000) prepared 30 minutes in advance in ' 1X PBS buffer containing 10% goat serum.
The slides are then incubated for 30 minutes in a humid atmosphere (assured by a filter soaked in PBS at the bottom of the box in which is deposited the blade), at room temperature, then rinsed once in 1X PBS at is room temperature (in the dark).
The slides are then washed in 3 baths of PBS 1 X, 5 min each, under stirring (in the dark), and are preserved if necessary at 4 ° C in a 1X PBS bath (in the dark).
Example 5 - Reading step 2o A. On the slides in radioactive mode The reading is done with the micro-imager (Biospace), the blade having been covered with a solid scintillating leaf.
B. On slides in chemiluminescent mode A nylon membrane (Hybond N +) with a thickness of 50 μm and a white appearance, as impregnated with CDPStar substrate (Amersham) diluted 100 times in the buffer substrate dilution (Roche ref 1759 779) is added to the slide. The reading is then done with the micro-imager (Biospace).
Example 6- Results A. Identification of the four model agents 106 cells from each of the four agents were treated according to protocol presented above. The sample probes were prepared following the mode of radioactive labeling then hybridized on the chips to DNA. The images below correspond to those obtained after 10 minutes of acquisition on the micro-imager.
The spots allowing Figure 3 to identify the four agents are circled in red for the ARN16S capture probes and in blue for the MRNA.
The summary tables below represent the pattern of hybridization corresponding to each of the model agents.

.a, z p H

C H ' O
E-O

c4 N

aao acted Rf N

at. ao cn ~, acted z NOT

CV O. '~, ..
Z
VS

N cfl your f "'N

ZN H ' CG

O
CH

O
H

M

f ~ O
p G7 ~ CD
T

E ... M
.

CV .C
~ CC ~
CD

+ _ O
Z
O I- Z

V

W
ZO
i-, Z

Results show that E. coli sample probes hybridize specifically with the oligonucleotides of E. coli and P.
agglomerans. However, we notice that the intensity of the spots is more strong for both E. coli16S RNAs and P. agglomerans16S RNAs.
s Indeed, the average ratio between the intensity of the 16Scoli spots and that of 16SPa spots is 2.12.
Non-specific hybridizations are observed as for the specific 16SBth3 16SBth2 oligonucleotide of B. fhuringiensis and the specific oligonucleotide RecA2 of 8. globigü. However these 1o unspecific hybridizations are also observed for all the others agents.

U
H

~

_ UH

o I-O
H

tSt N

a Q o cn co ~ c Rf N

a Q o ' r ~ C

O

~ ~

O
m i- 'd VS

BOY WUT

QM
Z
-i- ~ n E "' H
T

G ~

t c ~ r O
, y Z

O
VS
-M

O

tfl C
~
T

M

O
V.4 C / ~

O
O
VS

O
O
VS

The results show that the sample probes of P. agglomerans not only hybridize with the 16S RNAs of P. agglomerans and E.
coli but also with B.suburiensis, 16SBth2 and 16SBth316SRNAs.
The average ratio of spot intensities between 16SPa and 16Scoli is s 1.13 showing that spot intensity is more intense for 16S RNAs of P. agglomerans only for the 16S RNA of E. coli.
Specific mRNA for P. agglomerans is detected. This is Crth1 which encodes a protein involved in the biosynthesis of ~ i-carotene.
Non-specific hybridizations are observed as for The oligonucleotide 16SBth3 16SBth2 specific for B. thuringiensis and the oligonucleotide RecA2 specific for B. globigü. However these non-specific hybridizations are also observed for all the others agents.

'y -vs '' t--_ o VS
-O

OC

O

d O

O
1-, VS

O
O
Z

NN
t_ Z
- ~ 00 m O tn ~ H
r O
NZ
m NOT

rr t cG

O
t_ Z
'C dm CH

~

T

M
NOT

cfl ,Gold M

N ~

_ _ Cn O CC

r VS

O
, O
VS

m O
NOT
O

The results show that B. thuringiensis specific 16SRNAs are visible, 16SBthl, 16SBth2, 16SBth3. Other non-oligonucleotides specific are not revealed. The 16SBS2 oligonucleotide specific for 8.
globigü is detected in the presence of sample probes of 8.
thuringiensis. This result is explained by the presence of 10 mismatches between the 16S16 thuringiensis RNA and the oligonucleotide 16SBS2 specific of 8. globigü. Our hybridization protocol does not allow not discriminate below 10 mismatches.
Here, only the 16SBth1 capture probe can identify B. thuringiensis.
Indeed, the signals corresponding to 16SBth2 and 16SBth3 are visible for all agents H

O

H

O

m Z
' N I_ VS
O

Z

u7 F-O

A

VS

O

C7 'N

O

'd VS

O

i-NOT

NN

t _ '00 m Z

C (~ ~ H

r T

NN

fn f / ~! -r T

O

it has d E- M

O

NOT

~

O

'd r' r M

s 'm C (n NOT

LZ

. ~

NOT

O

VS

The results show that the B16-specific B16 globigues are visible 16SBS1 and 16SBS2. Only the 16SBS1 capture probe allows to identify B. globigü.
In summary, the specific probes of 16S RNAs make it possible to differentiate s all the agents between them. Comparison of intensity values of signals obtained makes it possible to discriminate between P, agglomerans and E. coli. In Indeed, E. soli is identified for a l6Scoli / 16SPa ratio greater than 1 and P.
agglomerans for a rapoort less than 1. In addition the detection of mRNA
Crth, specific for P. agglomerans confirms the analysis based on the detection of 16S RNAs.

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Claims (20)

A method of rapid detection of microorganisms, comprising:

at. the collection of microorganisms, b. the extraction of RNA from microorganisms, vs. RNA labeling or conversion to complementary DNA
Mark, d. hybridization of labeled complementary DNA or RNA
labeled obtained in step (c) using capture probes placed on a chip and specific to each micro-organism, characterized in that the hybridization is carried out in a time of between 15 and 45 minutes, preferably about 30 minutes, e. detection of possible hybridization, and, f. analysis of the results to determine whether the micro-organisms are present. 2. Method according to claim 1, characterized in that the DNA or the RNA is radioactively labeled. 3. Method according to claim 1, characterized in that the DNA or RNA is labeled with a chemiluminescent label. 4. Method according to claim 2, characterized in that the radioactive marker is the S35. 5. Method according to claim 3, characterized in that the marker is a biotin / streptavidin / biotin enzymatic complex. 6. Method according to claim 1, characterized in that before step (b), the microorganisms are subjected to a step of revivification.

42. 7. Method according to claim 6, characterized in that the step of revivification includes the culture of microorganisms in the middle brain infusion. 8. Method according to one of claims 1 to 7, characterized in that that the RNA is extracted after lysis of the microorganisms. 9. Method according to any one of claims 1 to 8, characterized in that the chip is a glass slide covered with polylysine silane or epoxy. 10. Method according to claim 9, characterized in that the epoxy is consisting of 3-glycindoxypropyl trimethoxy silane. 11. Method according to any one of claims 1 to 10, characterized in that the capture probe is of a length between 30 and 90 nucleotides. 12. Method according to any one of claims 1 to 11, characterized in that the capture probe is of a length about 70 nucleotides. 13. Method according to any one of claims 1 to 12, characterized in that the microorganisms are selected in the next group:
Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus luteus, Micrococcus roseus, Lactococcus lactis, Streptococcus pneumoniae, Streptococcus bovis, Leuconostoc mesenteroides, Oenococcus oeni, Pediococcus damnosus, Enterococcus durans, Enterococcus Fecalis, Enterococcus faecium, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Acinetobacter baumannii, Acinetobacter Iwoffii, Citrobacter diversus, Citrobacter freundii, Various citrobacter, Edwardsiella tarda, Enterobacter cloacae, Erwinia chrysantum, Erwinia carotovora, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, Morganella morganii, Pantoea agglomerans, Proteus mirabilis, Proteus vulgaris, Providencia alkalifaciens, Salmonella enterica subsp. enterica, Salmonella enterica, Salmonella paratyphiSerratia marcescens, Shigella flexneri, Yersinia enterocolitica, Yersinia pestis, Vibrio parahaemolyticus, Vibrio cholerae, Aeromonas hydrophila, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei, Ralstonia pickettii, Alcaligenes faecalis, Acinetobacter baumannii, Acinetobacter Iwoffii, Stenotrophomonas maltophilia, Campylobacter jejuni, Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, Bacillus anthracis, Bacillus globigii, Listeria monocytogenes, Lactobacillus casei, Clostridium botulinum, Clostridium perfringens, Mycobacterium bovis BCG, Legionella pneumophila, Bordetella pertussis, Francisella tularensis, Brucella Melitensis, Brutella abortus, Brucella suis, Brucella canisCoxiella burnetti, Chlamydia pneumonia. 14. Method according to any one of claims 1 to 13, characterized in that the microorganisms are collected using an air sampler. 15. Method according to claim 2 or 4, characterized in that the implementation time is about 2 1/2 hours. 16. Method according to claim 3 or 5, characterized in that the implementation time is about 3 hours. 17. Method for decreasing the scattering of light on a microchip, said method comprising:

at. the preparation of a chip on the surface of which is deposited a porous membrane impregnated with a substrate chemiluminescent, thus immobilizing the liquid phase of the substrate. 18. Method according to claim 17, characterized in that the porous membrane is nylon. 19. Chip suitable for a chemiluminescence revelation, said chip comprising a thin layer of glass on which is deposited microorganism-specific capture probes and a porous membrane impregnated with a chemiluminescent substrate. 20. Chip according to claim 19, characterized in that the membrane porous is nylon.